Liquid chromatography is a chromatographic technique used to separate components of a mixture based on a particular characteristic of the components. Liquid chromatography is used to identify each component in a mixture and to quantify each component. In general, liquid chromatography involves a liquid sample being passed over a solid adsorbent material packed into a column using a flow of liquid solvent. Each component (e.g., analyte) in the sample interacts slightly differently with the adsorbent material, thus retarding the flow of the analytes. If the interaction is weak, the analytes flow off the column in a short amount of time, and if the interaction is strong, then the analytes take a longer time to flow off the column.
The active component of the column, referred to as the sorbent, is typically a granular material made of solid particles (e.g., silica, polymers, etc.), and can range from approximately 2-50 micrometers in size. The components of the sample mixture are separated from each other due to their different degrees of interaction with the sorbent particles. The pressurized liquid is typically a mixture of solvents (e.g. water, acetonitrile or methanol) and is referred to as a “mobile phase”. The composition and temperature of the mobile phase liquid plays a major role in the separation process by influencing the interactions between sample components and the sorbent. These interactions are physical in nature, such as hydrophobic (dispersive), dipole-dipole and ionic, or some combination thereof.
The sample mixture to be separated and analyzed is introduced, in a discrete small volume (typically microliters), into the stream of mobile phase percolating through the column. The components of the sample move through the column at different velocities, which are function of specific physical interactions with the sorbent (also called stationary phase). The velocity of each component depends on its chemical nature, on the nature of the stationary phase (column) and on the composition of the mobile phase. The time at which a specific analyte emerges from the column is referred to as its “retention time”. The retention time measured under particular conditions is considered an identifying characteristic of a given analyte.
A liquid chromatography system generally includes a sampler, pumps, and a detector. The sampler brings the sample mixture into the mobile phase stream which carries it into the column. The pumps deliver the desired flow and composition of the mobile phase liquid through the column. The detector generates a signal proportional to the amount of sample component emerging out of the column, thus allowing for quantitative analysis of the sample components. A general-purpose or special purpose digital computer can be configured to control the liquid chromatography system and to provide the data analysis. Various detectors in common use include UV detectors, photodiode arrays (“PDAs”), florescence detectors, or mass spectrometry-based detectors. External detectors may also be used (e.g., fluorescence, refractive index, etc.). Also many different types of columns are available, filled with sorbents varying in particle size.
FIG. 1 depicts an example graphical representation of a liquid chromatography system according to the prior art. In the illustrated embodiment, liquid chromatography unit 100 includes (1) solvent reservoirs, (2) solvent degasser, (3) gradient valve, (4) mixing vessel for delivery of the mobile phase, (5) high-pressure pump, (6) switching valve, (7) sample injection loop, (8) pre-column (guard column), (9) analytical column, (10) detector (e.g., IR or UV), (11) data processing apparatus, and (12) waste collector.